Abstract The host microenvironment is necessary for tumor growth and metastasis, and a major determinant of resistance to treatment and relapse. Expression of N-cadherin (Ncad), a calcium-dependent cell-cell adhesion molecule, in cancer associated fibroblasts (CAF) has been reported to favor tumor growth. Ncad is the main cadherin expressed in bone cells, where it functions in cell-cell adhesion, but also regulates signaling and differentiation. In preliminary studies we found that, contrary to expectations, ablation of the Ncad gene (Cdh2) in osteolineage cells ? expressing the osteogenic marker, Osterix (Osx+) ? does not affect bone engraftment of breast cancer cells; however, subcutaneous tumors grow faster and lung metastases develop earlier than in wild type littermates. We also find, unexpectedly, that Ncad is present in previously unrecognized Osx+ cells in extra-skeletal tumors. These cells have a transcriptomic profile more similar to osteogenic cells than to CAF, and favor tumor growth. Furthermore, Ncad in Osx+ cells down-regulates p38 responsive genes, a pro- tumorigenic pathway. In human breast cancer, Osx+ are an index of poor prognosis. These preliminary results demonstrate that Ncad in Osx+ cells is a negative regulator of cancer progression, an effect opposite to Ncad reported action in CAF. We contend that Ncad exerts multiple and even opposite actions on tumorigenesis depending on the cell context where it is expressed, via modulation of specific signaling pathways. Based on these preliminary data, our central hypothesis is that Ncad in pro-tumorigenic Osx+ cells restrains tumor growth by regulating signals that reprogram the tumor microenvironment. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1 ? Modulation of extra-skeletal tumor growth by Ncad in Osx+ cells; testing the hypothesis that Ncad in Osx+ cells restrains tumor growth; loss of Ncad in TAOC increases tumor growth and metastases in mice. Osx+ Ncad+ cells correlate with tumor grading in human breast cancer. Specific Aim 2 ? Mechanisms of Ncad modulation of pro-tumorigenic signals in tumor-associate Osx+ cells; testing the hypothesis that Ncad in Osx+ cells is an upstream regulator of p38 and Pten signaling; loss of Ncad in Osx+ cells results in accentuated expression of p38-dependent pro- tumorigenic factors and decreased Pten dependent signals, leading to tumor microenvironment modification and enhanced tumorigenesis. We will use in vivo approaches, including diphtheria toxin-induced selective cell ablation, parabiosis, lineage tracking, as well as non-biased transcriptomic approaches (single cell RNAseq) to unlock the cellular and molecular mechanisms by which Ncad in extraskeletal Osx+ cells affects tumor growth and metastasis. We will also determine the clinical pathology correlates of Ncad expression in Osx+ cells in human tumors. Results of the proposed studies will lay the foundations for the development of new markers of tumor progression and/or new therapeutic strategies aimed at interrupting environmental support of cancer growth and metastasis by targeting specific cells in the tumor stroma.